Progression of Kidney Disease in Kidney Transplant Recipients With a Failing Graft: A Matched Cohort Study.

Background: Few studies have assessed outcomes in transplant recipients with failing grafts as most studies have focused on outcomes after graft loss. Objective: To determine whether renal function declines faster in kidney transplant recipients with a failing graft than in people with chronic kidney disease of their native kidneys. Design: Retrospective cohort study. Setting: Alberta, Canada (2002-2019). Patients: We identified kidney transplant recipients with a failing graft (2 estimated glomerular filtration rate [eGFR] measurements 15-30 mL/min/1.73 m2 ≥90 days apart). Measurements: We compared the change in eGFR over time (eGFR with 95% confidence limits, LCLeGFRUCL) and the competing risks of kidney failure and death (cause-specific hazard ratios [HRs], LCLHRUCL). Methods: Recipients (n = 575) were compared with propensity-score-matched, nontransplant controls (n = 575) with a similar degree of kidney dysfunction. Results: The median potential follow-up time was 7.8 years (interquartile range, 3.6-12.1). The hazards for kidney failure (HR1.101.331.60) and death (HR1.211.592.07) were significantly higher for recipients, while the eGFR decline over time was similar (recipients vs controls: -2.60-2.27-1.94 vs -2.52-2.21-1.90 mL/min/1.73 m2 per year). The rate of eGFR decline was associated with kidney failure but not death. Limitations: This was a retrospective, observational study, and there is a risk of bias due to residual confounding. Conclusions: Although eGFR declines at a similar rate in transplant recipients as in nontransplant controls, recipients have a higher risk of kidney failure and death. Studies are needed to identify preventive measures to improve outcomes in transplant recipients with a failing graft.

Contexte: Peu d'études ont évalué les résultats chez les patients transplantés dont le greffon est défaillant; la majorité des études s'étant plutôt concentrées sur les résultats après la perte du greffon. Objectif: Vérifier si la fonction rénale décline plus rapidement chez les patients transplantés dont le greffon est défaillant que chez les personnes souffrant d'une insuffisance chronique sur reins natifs. Conception: Étude de cohorte rétrospective. Cadre: Alberta, Canada (2002 à 2019). Sujets: Nous avons identifié des patients transplantés dont le greffon est défaillant (défini par deux mesures du débit de filtration glomérulaire estimé [DFGe] de 15-30 ml/min/1,73 m2 à au moins 90 jours d'intervalle). Mesures: Nous avons comparé l'évolution du DFGe dans le temps (DFGe avec intervalles de confiance [IC] à 95 % inférieur et supérieur: ICIDFGeICS) et les rapports de risque d'insuffisance rénale et de décès (intervalles de rapport de risque (RR) lié à la cause: ICIRRICS). Méthodologie: Les transplantés dont le greffon est défaillant (n=575) ont été comparés à des témoins non transplantés (n=575) appariés selon le score de propension et présentant un niveau similaire de dysfonctionnement rénal. Résultats: Le temps médian de suivi potentiel était de 7,8 ans (ÉIQ: 3,6 à 12,1). Les risques d'insuffisance rénale (RR: 1,101,331,60) et de décès (RR: 1,211,592,07) étaient significativement plus élevés chez les transplantés dont le greffon est défaillant, mais le déclin du DFGe au fil du temps était similaire dans les deux groupes (receveurs: -2,60-2,27-1,94 ml/min/1,73 m2 par an; témoins: -2,52-2,21-1,90 ml/min/1,73 m2 par an). Le taux de déclin du DFGe a été associé à une insuffisance rénale terminale, mais pas au décès. Limites: Il s'agit d'une étude observationnelle rétrospective et il existe un risque de biais dû à des facteurs de confusion résiduels. Conclusion: Bien que le DFGe décline à un rythme similaire chez les transplantés dont le greffon est défaillant et les témoins non transplantés, le risque d'insuffisance rénale terminale et de décès est plus élevé pour les transplantés. D'autres études sont nécessaires pour identifier les mesures préventives qui pourraient améliorer les résultats des patients transplantés dont le greffon est défaillant.

Differences in medication adherence by sex and organ type among adolescent and young adult solid organ transplant recipients.

BACKGROUND: Identification of differences in medication adherence by sex or organ type may help in planning interventions to optimize outcomes. We compared immunosuppressive medication adherence between males and females, and between kidney, liver and heart transplant recipients. METHODS: This multicenter study of prevalent kidney, liver and heart transplant recipients 14-25 years assessed adherence 3 times (0, 3, 6 months post-enrollment) with the BAASIS self-report tool. At each visit, participants were classified as adherent if they missed no doses in the prior 4 weeks and non-adherent otherwise. Adherence was also assessed using the coefficient of variation (CV) of tacrolimus trough levels; CV < 30% was classified as adherent. We used multivariable mixed effects logistic regression models adjusted for potential confounders to compare adherence by sex and by organ. RESULTS: Across all visits, males (n = 150, median age 20.4 years, IQR 17.2-23.3) had lower odds of self-reported adherence than females (n = 120, median age 19.8 years, IQR 17.1-22.7) (OR 0.41, 95% CI 0.21-0.80) but higher odds of adherence by tacrolimus CV (OR 2.50, 95% CI 1.30-4.82). No significant differences in adherence (by self-report or tacrolimus CV) were noted between the 184 kidney, 58 liver, and 28 heart recipients. CONCLUSION: Females show better self-reported adherence than males but greater variability in tacrolimus levels. Social desirability bias, more common in females than males, may contribute to better self-reported adherence among females. Higher tacrolimus variability among females may reflect biologic differences in tacrolimus metabolism between males and females rather than sex differences in adherence. There were no significant differences in adherence by organ type.

Premortem anticoagulation timing and dose in donation after circulatory death: multicentre study of associations with graft function.

BACKGROUND: In controlled donation after circulatory determination of death (DCD), it is common to administer premortem heparin to potential donors. This practice remains controversial because there is limited evidence for it and there is the possibility of inducing hemorrhage. To our knowledge, no previous studies have assessed the effects of heparin timing and dose on graft function. METHODS: We performed a multicentre cohort study of consecutive DCD donors and the recipients of their organs. Anticoagulation administration was considered early if given near the time of withdrawal of life-sustaining measures and late if delayed until the onset of donor hypoxemia (oxygen saturation < 70%) or hypotension (systolic blood pressure < 60 mm Hg or mean blood pressure < 50 mm Hg). The anticoagulation dose was considered high if it was 300 units/kg or greater. RESULTS: Donor anticoagulation data were available for 301 kidney, 75 liver and 46 lung recipients. Heparin was administered in 92% of cases and was most commonly withheld in donors with cerebrovascular causes of death (p = 0.01). Administration was late in 59% and the dose was low in 27%. Among kidney recipients, there were no significant differences in need for dialysis, glomerular filtration rate over the first year after transplantation or graft survival on the basis of whether or not the donor received heparin, the timing of heparin administration or the dose of heparin. Among liver recipients, alkaline phosphatase concentrations over the first year were significantly higher among recipients who received organs from donors to whom lower doses of heparin had been administered. CONCLUSION: Premortem heparin is widely used in DCD cases, but there is variability in timing and dose, which was not associated with kidney outcomes in this study. Donor anticoagulation may have a greater impact in preventing biliary complications following liver transplantation.

Immunosuppressant Medication Use in Patients with Kidney Allograft Failure: A Prospective Multicenter Canadian Cohort Study.

BACKGROUND: Patients with kidney transplant failure have a high risk of hospitalization and death due to infection. The optimal use of immunosuppressants after transplant failure remains uncertain and clinical practice varies widely. METHODS: This prospective cohort study enrolled patients within 21 days of starting dialysis after transplant failure in 16 Canadian centers. Immunosuppressant medication use, death, hospitalized infection, rejection of the failed allograft, and anti-HLA panel reactive antibodies were determined at 1, 3, 6, and 12 months and and then twice yearly until death, repeat transplantation, or loss to follow-up. RESULTS: The 269 study patients were followed for a median of 558 days. There were 33 deaths, 143 patients hospitalized for infection, and 21 rejections. Most patients (65%) continued immunosuppressants, 20% continued prednisone only, and 15% discontinued all immunosuppressants. In multivariable models, patients who continued immunosuppressants had a lower risk of death (hazard ratio [HR], 0.40; 95% confidence interval [CI], 0.17 to 0.93) and were not at increased risk of hospitalized infection (HR, 1.81; 95% CI, 0.82 to 4.0) compared with patients who discontinued all immunosuppressants or continued prednisone only. The mean class I and class II panel reactive antibodies increased from 11% to 27% and from 25% to 47%, respectively, but did not differ by immunosuppressant use. Continuation of immunosuppressants was not protective of rejection of the failed allograft (HR, 0.81; 95% CI, 0.22 to 2.94). CONCLUSIONS: Prolonged use of immunosuppressants >1 year after transplant failure was not associated with a higher risk of death or hospitalized infection but was insufficient to prevent higher anti-HLA antibodies or rejection of the failed allograft.

Acute Kidney Allograft Rejection Following Coronavirus mRNA Vaccination: A Case Report.

Supplemental Digital Content is available in the text.

Care processes and structures associated with higher medication adherence in adolescent and young adult transplant recipients.

BACKGROUND: We aimed to identify care processes and structures that were independently associated with higher medication adherence among young transplant recipients. METHODS: We conducted a prospective, observational cohort study of 270 prevalent kidney, liver, and heart transplant recipients 14-25 years old. Patients were ≥3 months post-transplant, ≥2 months post-discharge, and followed in one of 14 pediatric or 14 adult transplant programs in Canada. Patients were enrolled between June 2015 and March 2018 and followed for 6 months. Adherence was assessed at baseline, 3, and 6 months using the BAASIS© self-report tool. Patients were classified as adherent if no doses were missed in the prior 4 weeks. Transplant program directors and nurses completed questionnaires regarding care organization and processes. RESULTS: Of the 270 participants, 99 were followed in pediatric programs and 171 in adult programs. Median age was 20.3 years, and median time since transplant was 5 years. At baseline, 71.5% were adherent. Multivariable mixed effects logistic regression models with program as a random effect identified two program-level factors as independently associated with better adherence: minimum number of prescribed blood draws per year for those >3 years post-transplant (per 1 additional) (OR 1.12 [95% CI 1.00, 1.26]; p = .047), and average time nurses spend with patients in clinic (per 5 additional minutes) (OR 1.15 [1.03, 1.29]; p = .017). CONCLUSION: Program-level factors including protocols with a greater frequency of routine blood testing and more nurse time with patients were associated with better medication adherence. This suggests that interventions at the program level may support better adherence.

Successful Treatment of a Reinfected Liver Graft Because of Receipt of a HCV-Positive Kidney.

Transplantation of hepatitis C virus (HCV)-positive organs has undergone a paradigm shift because of the advent of direct-acting antivirals. We present the case of a 57-year-old man successfully treated initially with pegylated interferon and ribavirin after HCV recurrence postliver transplantation. He subsequently developed end-stage renal disease and received a genotype 1a HCV-positive kidney transplant. A 12-week course of ledipasvir/sofosbuvir and low-dose ribavirin was initiated and sustained virologic response was achieved. This constitutes the first reported case of a patient successfully treated for HCV a second time after receiving an HCV-positive organ.

Mortality and Morbidity in Kidney Transplant Recipients With a Failing Graft: A Matched Cohort Study.

BACKGROUND: Due to their history of renal disease and exposure to immunosuppression, kidney transplant recipients with a failing graft may be at higher risk of adverse outcomes compared to nontransplant controls. Understanding the burden of disease in transplant recipients may inform treatment decisions of people whose native kidneys are failing and may be eligible for a transplant. OBJECTIVE: To compare mortality and morbidity in kidney transplant recipients with a failing graft to matched nontransplant controls. DESIGN: Retrospective cohort study. SETTING: Alberta, Canada. PATIENTS: Kidney transplant recipients with a failing graft were identified as having at least 2 estimated glomerular filtration rate (eGFR) measurements between 15-30 mL/min/1.73 m2 (90-365 days apart). We also identified nontransplant controls with a similar degree of kidney dysfunction. MEASUREMENTS: Mortality and hospitalization. METHODS: We propensity-score matched 520 kidney transplant recipients with a failing graft to 520 nontransplant controls. RESULTS: The median age of the matched cohort was 57 years and 40% were women. Compared to matched nontransplant controls, recipients with a failing graft had a higher hazard of death (hazard ratio, 1.54; 95% confidence interval [CI], 1.28-1.85; p < .001) and a higher rate of all-cause hospitalization (rate ratio, 1.67; 95% CI, 1.42-1.97; p < .001). Kidney transplant recipients also had a higher rate of several cause-specific hospitalizations including genitourinary, cardiovascular, and infectious causes. LIMITATIONS: Observational design with the risk of residual confounding. CONCLUSIONS: A failing kidney transplant is associated with an increased burden of mortality and morbidity beyond chronic kidney disease. This information may assist the discussion of prognosis in kidney transplant recipients with a failing graft and the design of strategies to minimize risks.

CONTEXTE: En raison de leurs antécédents de néphropathie et de leur exposition aux immunosuppresseurs, les receveurs d'une greffe rénale dont le greffon est défaillant pourraient être plus susceptibles de souffrir de pathologies associées que les patients non transplantés (contrôles). Comprendre le fardeau de la maladie pour les receveurs d'une greffe pourrait orienter les décisions de traitement pour les patients dont les reins natifs sont défaillants et qui sont admissibles à une greffe. OBJECTIF: Comparer la mortalité et les comorbidités de receveurs d'une greffe rénale dont le greffon est défaillant à celles de patients non greffés (contrôles). TYPE D'ÉTUDE: Étude de cohorte rétrospective. CADRE: Alberta, Canada. SUJETS: Le statut de receveur avec greffon défaillant a été établi par au moins deux mesures de DFGe se situant entre 15 et 30 ml/min/1.73 m2 (de 90 à 365 jours d'intervalle). Des patients non greffés présentant un dysfonctionnement rénal similaire ont servi de contrôles. MESURES: Mortalité et nombre d'hospitalisations. MÉTHODOLOGIE: Nous avons jumelé 520 receveurs avec greffon défaillant à 520 patients non greffés sur la base du score de propension. RÉSULTATS: L'âge médian des sujets était de 57 ans et 40 % étaient des femmes. Les patients avec un greffon défaillant ont présenté un risque de mortalité (rapport de risque : 1.54; IC 95 % : 1.28-1.85; p < .001) et un taux d'hospitalization toutes causes confondues (rapport des taux : 1.67; IC 95%, 1.42-1.97; p < .001) plus élevés que les patients non greffés. Ils étaient également hospitalisés plus fréquemment, notamment pour des problèmes génito-urinaires ou cardiovasculaires, ou pour des infections. LIMITES: La nature observationnelle de l'étude pourrait comporter des facteurs de confusion résiduels. CONCLUSION: Une transplantation rénale défaillante a été associée à un plus grand risque de morbidité et de mortalité que l'insuffisance rénale chronique. Cette information pourrait orienter les discussions concernant le pronostic des receveurs d'un rein dont le greffon est défaillant et guider l'élaboration de stratégies pour minimiser les risques.

Donation after circulatory determination of death in western Canada: a multicentre study of donor characteristics and critical care practices.

PURPOSE: Donation after circulatory determination of death (DCD) has been performed in Canada since 2006. Numerous aspects of donor management remain controversial. METHODS: We performed a multicentre cohort study involving potential DCD donors in western Canada (2008-2017), as well as recipients of their organs, to describe donor characteristics and critical care practices, and their relation to one-year recipient and graft survival. RESULTS: There were 257 patients in four provinces that underwent withdrawal of life-sustaining therapies (WLST) in anticipation of possible DCD. The proportion of patients that died within two hours of WLST ranged from 67% to 88% across provinces (P = 0.06), and was predicted by deeper coma (P = 0.01), loss of pupillary light or corneal reflexes (P = 0.02), and vasopressor use (P = 0.01). There were significant differences between provinces in time intervals from onset of hypotension to death (9-11 min; P = 0.02) and death to vascular cannulation (7-10 min; P < 0.001). There was inconsistency in pre-mortem heparin administration (82-96%; P = 0.03), including timing (before vs after WLST; P < 0.001) and dose (≥ 300 vs < 300 units·kg-1; P < 0.001). Donation after circulatory death provided organs for 321 kidney, 81 liver, and 50 lung transplants. One-year recipient and graft survival did not differ among provinces (range 85-90%, P = 0.45). Predictors of death or graft failure included older recipient age (odds ratio [OR] per year, 1.04; 95% confidence interval [CI],1.01 to 1.07) and male donor sex (OR, 3.35; 95% CI, 1.39 to 8.09), but not time intervals between WLST and cannulation or practices related to heparin use. CONCLUSION: There is significant variability in critical care DCD practices in western Canada, but this has not resulted in significant differences in recipient or graft survival. Further research is required to guide optimal management of potential DCD donors.

RéSUMé: OBJECTIF: Le don d'organes après décès cardiocirculatoire (DDC) est pratiqué au Canada depuis 2006. De nombreux aspects touchant à la prise en charge des donneurs demeurent controversés. MéTHODE: Nous avons réalisé une étude de cohorte multicentrique auprès de donneurs potentiels de DDC dans l'Ouest canadien (2008­2017), ainsi qu'auprès des récipiendaires de leurs organes, afin de décrire les caractéristiques des donneurs et les pratiques de soins intensifs, ainsi que la relation entre ces éléments et la survie à un an des récipiendaires et des organes greffés. RéSULTATS: Au total, 257 patients provenant de quatre provinces ont subi une interruption des traitements de survie en vue d'un possible DDC. La proportion de patients décédés dans les deux heures suivant l'interruption des traitements de survie allait de 67 % à 88 % dans toutes les provinces à l'étude (P = 0,06) et pouvait être prédite par une profondeur du coma plus importante (P = 0,01), la perte de la réaction pupillaire à la lumière ou des réflexes cornéens (P = 0,02), et l'utilisation de vasopresseurs (P = 0,01). Des différences significatives ont été observées entre les différentes provinces dans les intervalles de temps entre le début de l'hypotension et le décès (9­11 min; P = 0,02) et entre le décès et la canulation vasculaire (7­10 min; P < 0,001). Il y avait divergence dans l'administration d'héparine avant le décès (82-96 %; P = 0,03), notamment en ce qui concerne le moment d'administration (avant vs après l'interruption des traitements de survie; P < 0,001) et la posologie (≥ 300 vs < 300 unités·kg−1; P < 0,001). Le don après décès cardiocirculatoire a permis de procurer des organes pour 321 greffes rénales, 81 greffes hépatiques et 50 greffes pulmonaires. La survie à un an du récipiendaire et du greffon ne différait pas d'une province à l'autre (allant de 85 à 90 %, P = 0,45). Les prédicteurs de décès ou de la défaillance du greffon incluaient l'âge plus avancé du récipiendaire (rapport de cotes [RC] par année, 1,04; intervalle de confiance [IC] 95 %, 1,01 à 1,07) et un donneur de sexe masculin (RC, 3,35; IC 95 %, 1,39 à 8,09), mais pas les intervalles de temps entre l'interruption des traitements de survie et la canulation, ni les pratiques liées à l'utilisation d'héparine. CONCLUSION: Il existe une importante variabilité dans les pratiques de soins intensifs pour le DDC dans l'Ouest canadien, mais cette variabilité n'a pas résulté en différences significatives en matière de survie des récipiendaires ou des greffons. Des recherches supplémentaires sont nécessaires afin d'aiguiller la prise en charge optimale des donneurs potentiels de DDC.

Direct Effects of Empagliflozin on Extracellular Matrix Remodelling in Human Cardiac Myofibroblasts: Novel Translational Clues to Explain EMPA-REG OUTCOME Results.

BACKGROUND: Empagliflozin, an SGLT2 inhibitor, has shown remarkable reductions in cardiovascular mortality and heart failure admissions (EMPA-REG OUTCOME). However, the mechanism underlying the heart failure protective effects of empagliflozin remains largely unknown. Cardiac fibroblasts play an integral role in the progression of structural cardiac remodelling and heart failure, in part, by regulating extracellular matrix (ECM) homeostasis. The objective of this study was to determine if empagliflozin has a direct effect on human cardiac myofibroblast-mediated ECM remodelling. METHODS: Cardiac fibroblasts were isolated via explant culture from human atrial tissue obtained at open heart surgery. Collagen gel contraction assay was used to assess myofibroblast activity. Cell morphology and cell-mediated ECM remodelling was examined with the use of confocal microscopy. Gene expression of profibrotic markers was assessed with the use of reverse-transcription quantitative polymerase chain reaction. RESULTS: Empagliflozin significantly attenuated transforming growth factor ß1-induced fibroblast activation via collagen gel contraction after 72-hour exposure, with escalating concentrations (0.5 µmol/L, 1 µmol/L, and 5 µmol/L) resulting in greater attenuation. Morphologic assessment showed that myofibroblasts exposed to empagliflozin were smaller in size with shorter and fewer number of extensions, indicative of a more quiescent phenotype. Moreover, empagliflozin significantly attenuated cell-mediated ECM remodelling as measured by collagen fibre alignment index. Gene expression profiling revealed significant suppression of critical profibrotic markers by empagliflozin, including COL1A1, ACTA2, CTGF, FN1, and MMP-2. CONCLUSIONS: We provide novel data showing a direct effect of empagliflozin on human cardiac myofibroblast phenotype and function by attenuation of myofibroblast activity and cell-mediated collagen remodelling. These data provide critical insights into the profound effects of empagliflozin as noted in the EMPA-REG OUTCOME study.

BK Polyomavirus and the Transplanted Kidney: Immunopathology and Therapeutic Approaches.

BK polyomavirus is ubiquitous, with a seropositivity rate of over 75% in the adult population. Primary infection is thought to occur in the respiratory tract, but asymptomatic BK virus latency is established in the urothelium. In immunocompromised host, the virus can reactivate but rarely compromises kidney function except in renal grafts, where it causes a tubulointerstitial inflammatory response similar to acute rejection. Restoring host immunity against the virus is the cornerstone of treatment. This review covers the virus-intrinsic features, the posttransplant microenvironment as well as the host immune factors that underlie the pathophysiology of polyomavirus-associated nephropathy. Current and promising therapeutic approaches to treat or prevent this complication are discussed in relation to the complex immunopathology of this condition.

Ramipril versus placebo in kidney transplant patients with proteinuria: a multicentre, double-blind, randomised controlled trial.

BACKGROUND: Angiotensin-converting enzyme inhibitors have been shown to reduce the risk of end-stage renal disease and death in non-transplant patients with proteinuria. We examined whether ramipril would have a similar beneficial effect on important clinical outcomes in kidney transplant recipients with proteinuria. METHODS: In this double-blind, placebo-controlled, randomised trial, conducted at 14 centres in Canada and New Zealand, we enrolled adult renal transplant recipients at least 3-months post-transplant with an estimated glomerular filtration rate (GFR) of 20 mL/min/1·73m(2) or greater and proteinuria 0·2 g per day or greater and randomly assigned them to receive either ramipril (5 mg orally twice daily) or placebo for up to 4 years. Patients completing the final 4-year study visit were invited to participate in a trial extension phase. Treatment was assigned by centrally generated randomisation with permuted variable blocks of 2 and 4, stratified by centre and estimated GFR (above or below 40 mL/min/1·73 m(2)). The primary outcome was a composite consisting of doubling of serum creatinine, end-stage renal disease, or death in the intention-to-treat population. The principal secondary outcome was the change in measured GFR. We ascertained whether any component of the primary outcome had occurred at each study visit (1 month and 6 months post-randomisation, then every 6 months thereafter). This trial is registered with ISRCTN, number 78129473. FINDINGS: Between Aug 23, 2006, and March 28, 2012, 213 patients were randomised. 109 were allocated to placebo and 104 were allocated to ramipril, of whom 109 patients in the placebo group and 103 patients in the ramipril group were analysed and the trial is now complete. The intention to treat population (placebo n=109, ramipril n=103) was used for the primary analysis and the trial extension phase analysis. The primary outcome occurred in 19 (17%) of 109 patients in the placebo group and 14 (14%) of 103 patients in the ramipril group (hazard ratio [HR] 0·76 [95% CI 0·38-1·51]; absolute risk difference -3·8% [95% CI -13·6 to 6·1]). With extended follow-up (mean 48 months), the primary outcome occurred in 27 patients (25%) in the placebo group and 25 (24%) patients in the ramipril group (HR 0·96 [95% CI 0·55-1·65]); absolute risk difference: -0·5% (95% CI -12·0 to 11·1). There was no significant difference in the rate of measured GFR decline between the two groups (mean difference per 6-month interval: -0·16 mL/min/1·73m(2) (SE 0·24); p=0·49). 14 (14%) of patients died in the ramipril group and 11 (10%) in the placebo group, but the difference between groups was not statistically significant (HR 1·45 [95% CI 0·66 to 3·21]). Adverse events were more common in the ramipril group (39 [38%]) than in the placebo group (24 [22%]; p=0·02). INTERPRETATION: Treatment with ramipril compared with placebo did not lead to a significant reduction in doubling of serum creatinine, end-stage renal disease, or death in kidney transplant recipients with proteinuria. These results do not support the use of angiotensin-converting enzyme inhibitors with the goal of improving clinical outcomes in this population. FUNDING: Canadian Institutes of Health Research.

Anti-BK virus mechanisms of sirolimus and leflunomide alone and in combination: toward a new therapy for BK virus infection.

BACKGROUND: Human BK polyomavirus is the causative agent of BK nephropathy which is now the leading cause of early renal graft loss. Although no randomized clinical trials have supported this therapy, reduction of immunosuppressive drugs is the current BK nephropathy treatment. We hypothesized that inhibition of the intracellular protein kinase pathways activated by BK virus may be a more effective therapeutic strategy than reduction of immunosuppression. METHODS AND RESULTS: Four days after infection of renal epithelial cells lines CCD1103, CCD1105 and human primary tubular epithelial cells with BK virus, we found increased phosphorylation of 3'-phosphoinositide-dependent kinase-1 (PDK-1), the protein kinase Akt (Akt), mammalian target of rapamycin (mTOR), and 70 kDa ribosomal protein S6 kinase (p70S6K). To inhibit this pathway, we used sirolimus, which repressed p70S6K phosphorylation and reduced BK virus large T antigen expression in a dose-dependent manner. We then used the tyrosine kinase inhibitor leflunomide (using the active metabolite A77 1726), which decreased PDK1 and Akt phosphorylation and inhibited BK virus genome replication and early gene expression. The combination of sirolimus and leflunomide inhibited BK virus genome replication, large T antigen expression, PDK1, Akt, mammalian target of rapamycin, and p70S6K phosphorylation. CONCLUSIONS: On the basis of these results, we suggest that inhibition of protein kinase pathways with a combination of sirolimus and leflunomide may be an effective therapy for BK virus reactivation. Because both sirolimus and leflunomide possess immunosuppressive activity, combination therapy may reduce BK pathogenesis while maintaining appropriate transplant immunosuppression.

MAP kinase activation increases BK polyomavirus replication and facilitates viral propagation in vitro.

BK polyomavirus causes disease in immunosuppressed patients. BK virus replication was augmented in HEL-299 cells cultured in conditions that activated the MAP kinase, ERK1/2. To determine if MAP kinase activation increased BK virus replication, cells were treated with serum and phorbol 12-myristate 13-acetate (PMA). Serum and PMA stimulated large T-antigen expression and increased BK virus DNA replication. The effects of serum/PMA were directly related to MAP kinase signal activation since viral replication was reduced by the MEK1/2 inhibitor U0126. PMA also increased cyclin D1 expression and inhibition of cyclin D1/CDK4 complex and the cell cycle reduced BK virus infection. The PMA effect occurred independent of direct transcriptional activation of the viral NCCR. In HEL-299 cells, virus infection in high serum and PMA accelerated viral replication that resulted, within 7 days, in the production of high titer infectious BK virus. These results show that MAP kinase signal activation increases BK virus replication.

Mechanisms of human complement factor B induction in sepsis and inhibition by activated protein C.

To investigate the potential role of the local expression of alternative complement factor B (hBf) in human sepsis, we examined the induction of Bf gene expression in human peripheral blood monocytes (PBMCs) from patients with septic shock and the mechanisms of hBf gene regulation by tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and lipopolysaccharide (LPS) in human monocytes. PBMCs from septic shock patients showed increased hBf mRNA expression when compared with control patients. Costimulation with TNF-alpha and IFN-gamma or stimulation with LPS demonstrated a time- and dose-dependent induction of hBf mRNA expression in human PBMCs. A region of the hBf promoter between -735 and +128 bp was found to mediate IFN-gamma, TNF-alpha, and LPS responsiveness as well as the synergistic effect of IFN-gamma/TNF-alpha on hBf promoter activity. Site-directed mutagenesis of a IFN-gamma-activation site (GAS) cis element (-90 to -82 bp) abrogated IFN-gamma responsiveness. Mutagenesis of a nuclear factor (NF)-kappaB cis element at -466 to -456 bp abrogated TNF-alpha and LPS responsiveness of the Bf promoter. Thus hBf gene expression is induced in PBMCs from septic shock patients, and the induction of hBf by IFN-gamma, TNF-alpha, and LPS is through GAS and NF-kappaB cis-binding sites on the hBf promoter. Furthermore, activated protein C (APC) inhibited LPS-stimulated hBf promoter activity and protein expression in human monocytes suggesting that the beneficial effect of APC therapy in sepsis may in part be due to inhibition of complement induction and/or activation via the alternative pathway.

The Canadian ACE-inhibitor trial to improve renal outcomes and patient survival in kidney transplantation--study design.

BACKGROUND: In non-transplant patients with chronic kidney disease and proteinuria, inhibition of the renin-angiotensin system with an ACE-inhibitor or an angiotensin receptor blocker has been shown to delay the progression of renal disease. Observational studies in the kidney transplant population have produced conflicting results with some studies showing benefit and others no benefit of renin-angiotensin system blockade. METHODS: This report describes the design and methodological issues of a randomized controlled trial evaluating the effect of ramipril in a renal transplant population. This study has been funded by a peer-reviewed grant from the Canadian Institutes of Health Research and is registered on the International Standard Randomised Controlled Trial Number Registry (ISRCTN-78129473). RESULTS: The study will randomize 528 kidney transplant patients (11 Canadian centers) with proteinuria and an estimated GFR between 20 and 55 ml/min/1.73 m(2) to either ramipril (5 mg BID) or placebo. Patients, clinical staff and investigators will be blinded to treatment allocation. The primary outcome will be a composite measure incorporating doubling of serum creatinine, end stage renal disease or death. Principal secondary outcomes include: decline in GFR using a radioisotopic method, change in proteinuria, change in blood pressure, incidence of adverse events (e.g. hyperkalemia, anemia), incidence of cardiovascular events and health-related quality of life assessed by the Short Form-36 and the EuroQol-5D. CONCLUSIONS: Upon completion, this trial will provide clinically meaningful evidence about whether treatment with an ACE-inhibitor will reduce patient mortality and prolong allograft survival in renal transplant recipients.

ROS generation in endothelial hypoxia and reoxygenation stimulates MAP kinase signaling and kinase-dependent neutrophil recruitment.

Reactive oxygen species (ROS)-induced injury has been shown to occur during the reperfusion phase of ischemia-reperfusion and ROS are known to induce signaling events. We hypothesized that oxygen sensing in endothelial cells is also dependent on internal redox changes during hypoxia and that endothelial cells respond to changing oxygen environments via signaling, switching to an inflammatory phenotype. Endothelial cells exposed to relative hypoxia or the mitochondrial inhibitors rotenone, antimycin A, or FCCP show loss of mitochondrial membrane potential. During hypoxia, an increase in cytoplasmic ROS and glutathione S-transferase activity occurred, suggesting changes in intracellular redox state, mimicked with rotenone or FCCP but inhibited by antimycin A. Phosphorylation of stress-responsive mitogen-activated protein kinases occurred in hypoxia and was rapid and prolonged. Phosphorylation was inhibited by vitamin C, N-acetyl cysteine, or antimycin A. Chelation of intracellular calcium inhibits phosphorylation but the mitochondrial transition pore inhibitor cyclosporin A had no effect. Reoxygenation caused a further round of signaling, which was rapid but transient. Functionally, adhesion of neutrophils after hypoxia-reoxygenation under flow is ROS, P-selectin, and MAPK dependent. Therefore, changes in cellular signaling and phenotype are abrogated by ROS scavengers and suggest their use as therapeutic agents in ischemia-reperfusion.

Role of enteric glia in intestinal physiology: effects of the gliotoxin fluorocitrate on motor and secretory function.

The role of enteric glia in gastrointestinal physiology remains largely unexplored. We examined the actions of the gliotoxin fluorocitrate (FC) on intestinal motility, secretion, and inflammation after assessing its efficacy and specificity in vitro. FC (100 microM) caused a significant decrease in the phosphorylation of the glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diaz-4-yl)amino]-2-deoxyglucose in enteric glial cultures and a reduction in glial uptake of the fluorescent dipeptide Ala-Lys-7-amino-4-methylcoumarin-3-acetic acid in both the ileum and colon. Dipeptide uptake by resident murine macrophages or guinea pig myenteric neurons was unaffected by FC. Incubation of isolated guinea pig ileal segments with FC caused a specific and significant increase in glial expression of the phosphorylated form of ERK-1/2. Disruption of enteric glial function with FC in mice reduced small intestinal motility in vitro, including a significant decrease in basal tone and the amplitude of contractility in response to electrical field stimulation. Mice treated with 10 or 20 micromol/kg FC twice daily for 7 days demonstrated a concentration-dependent decrease in small intestinal transit. In contrast, no changes in colonic transit or ion transport in vitro were observed. There were no changes in glial or neuronal morphology, any signs of inflammation in the FC-treated mice, or any change in the number of myenteric nitric oxide synthase-expressing neurons. We conclude that FC treatment causes enteric glial dysfunction, without causing intestinal inflammation. Our data suggest that enteric glia are involved in the modulation of enteric neural circuits underlying the regulation of intestinal motility.